This invention relates to a mixed culture system of anaerobic thermophilic microorganisms and more particularly, to a mixed culture of the newly discovered thermophilic glycolytic anaerobe Thermoanaerobacter ethanolicus and Clostridium thermocellum. Further, the invention relates the novel process for producing ethanol from cellulose by fermentation in a nutrient medium with a mixed culture prepared from biologically pure cultures of Thermoanaerobacter ethanolicus and Clostridium thermocellum. The invention is the result of a contract with the U.S. Department of Energy.
Relatively few anaerobic microorganisms have been isolated and characterized that grow on carbohydrates (are glycolytic) and yield ethanol under thermophilic and extreme thermophilic conditions. Representative examples of well-characterized glycolytic anaerobic bacteria that will grow in a nutrient culture in the thermophilic to extreme thermophilic ranges belong to the genus Clostridium and include: C. thermoaceticum, C. tartarivorum, C. thermosaccharolyticum, C. thermocellum, C. thermocellulaseum, and C. thermohydrosulfuricum. Strains of the latter organism have been isolated and characterized by J. Wiegel and L. G. Ljungdahl (See Abstract I 75 of the Abstract of the Annual Meeting of the American Society of Microbiology, Las Vegas, Nev., USA, 1978 and J. Bacteriology, September 1979 in press). A neotype strain of C. thermohydrosulfuricum E 100-69 was isolated from the liquors of an Austrian sugar beet factory by F. Hollaus and U. Sleytr (See Arch. Mikrobiol. 86: 129-146, 1972).
In addition to these well known species, two non spore-forming strains representing new species of a new genus have been isolated and characterized. The newly discovered thermophilic anaerobes were isolated in biologically pure cultures and designated as Thermoanaerobacter ethanolicus. A representative stain of this new microorganism in a biologically pure subculture, designated JW 200, has been deposited in the patent strain collection of the American Type Culture Collection, Rockville, Md., USA. ATCC 31550 is the accession number assigned by the American Type Culture Collection to this strain. In the isolation, purification and characterization of this newly discovered species, which is not a Clostridium, it has been found that the new species is an efficient producer of ethanol from various carbohydrates, in particular, the most common mono- and di-saccharides.
Thermoanaerobacter ethanolicus has been disclosed and claimed in a copending application Ser. No. 74,287 filed on the same date by the same inventors. As disclosed in that application, T. ethanolicus is cultured in aqueous nutrient medium under anaerobic, thermophilic conditions and is used in a novel process for producing ethanol which comprises subjecting carbohydrates, particularly the saccharides, to fermentation action of the newly isolated microorganism T. ethanolicus in a biologically pure culture to form ethanol and recovering said ethanol. While T. ethanolicus efficiently ferments a variety of sugars to yield ethanol, one of the characteristics of the anaerobe is that it does not ferment cellulose. It is known that the anaerobic microorganism Clostridium thermocellum, isolated in 1950, ferments cellulose to hydrogen, carbon dioxide, ethanol, formate, acetate, lactate and to a lesser degree, dicarboxylic acids at thermophilic temperatures. (See McBee, R. H. 1950. The anaerobic thermophilic cellulolytic bacteria. Bacteriol. Rev. 14:51-63). However, it has been found that in the clostridial cellulase system produced of this fermentation, some of the products formed by the action of cellulase, such as cellobiose, and glucose tend to inhibit the growth of C. thermocellum thereby repressing the cellulase enzyme system. Other sugars possibly present in a fermentation medium like lactose may also inhibit cellulase. While C. thermocellum will utilize these sugars, growth is slow and the yield of products, such as ethanol, is small. Thus, C. thermocellum in biologically pure form, while useful for the break down of the cellulose, will not efficiently ferment the sugar substrate produced and that, in fact, the fermentation is inhibited by the substrate accumulation. A newly isolated representative strain of C. thermocellum designated JW 20 has been deposited in the patent strain collection of the American Type Culture Collection, Rockville, Md., USA. ATCC 31549 is the accession number assigned to this strain of C. thermocellum.
These microorganisms are, of course, of use for the possible anaerobic fermentation of various carbohydrates, such as the saccharides, and in mixed cultures to efficiently break down cellulose for the production of ethanol and other products of fermentation under thermophilic conditions. Yeast (Saccharomyces species) fermentation of sugar, as is well known, ordinarily must be conducted at less than about 37.degree. C. under semiaerobic conditions to yield ethanol. Further, the conditions must be carefully controlled to avoid contamination of harmful bacteria, fungi, and molds.